Gliederung

Objective

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic retinopathy, the serious vascular complication of diabetes, which was one of the major causes of adult blindness. The purpose of this study was to investigate the role of PKC on the alteration of endothelin-1, vascular endothelial growth factor (VEGF) in the cultured human umbilical vein endothelial cell (hUVEC).

Methods

The hUVEC line was cultured in DMEM with low glucose (5.5 mM) or high glucose (25 mM) respectively. The measurement of PKC activities from membranous and cytosolic fractions, VEGF protein were conducted by ELISA. The hUVEC line was analyzed for the expression of ET-1 and VEGF mRNA by semi-quantitative RT-PCR, and was also immunostained for ET-1 and VEGF by immunocytochemistry. The tight conjunction between the hUVEC was observed by transmission electron microscope (TEM). The effect of a general PKC inhibitor, GF109203X, on the alteration of ET-1 and VEGF was also observed in vitro.

Results

In the cultured HUVEC line, PKC specific activities from the membranous fraction were significantly increased by 45% in the media with high glucose (25 mM) compared with that in the media with low glucose (5.5 mM), and were reversed after exposure in the media with low glucose for an additional 48 h. The mRNA expression and immunoreactivity for VEGF and ET-1 were increased after exposure in the high glucose media, and were inhibited after addition of GF109203X (final concentration 10-5, 10-6, 10-7 M) in a dose-dependent manner. The impairment of the tight conjunction between the hUVEC could be obviously observed 48 h after exposure to the high glucose media and the integrity of the tight conjunction could be recovered for an additional 12 h culture with 10-5 M GF109203X.

Conclusions

The results from this study showed that high glucose could induce the activation of PKC, enhanced expression for ET-1 and VEGF, and the impairment of the tight conjunction in vitro. A general PKC inhibitor, GF109203X could reverse the upregulation of ET-1 and VEGF, and the integrity of the tight conjunction, which might be one of the key factors contributing to the pathogenesis of vascular complication of diabetes.